Systems known from the literature and from practice for combining fluorescence microscopy with FCS consist, for example, of a CLSM that is attached to an optical port of a microscope stand for the purpose of imaging, and of a separate illumination and detection unit for FCS that is attached to another port of the stand and serves to record and process the fluorescence signal at one point in each case (Zeiss). In a further implementation, fluorescence microscopy is effected via an epifluorescence setup and FCS measurement via a separate unit as just described (Brock, 1999). In a further implementation, imaging is effected using a CLSM, and the FCS excitation and detection unit via the same optic using the same (DKFZ) or separate detectors (Leica).
Intrinsic to all of the systems is that FCS measurement in particular may only be carried out at a single point on the sample at a time, and that the FCS measurement is carried out using conventional beam splitters and detection filters. The inherent, relatively long duration of an FCS measurement of at least a few seconds makes it very difficult to compare different measurement points with each other because living cells are analyzed in typical applications so that movements and structural changes occur within seconds and even faster and as a result well-defined positioning for different successive measurement points within the sample is difficult or impossible. The use, in particular, of the results of FCS analysis (as described above) as a contrast-providing signal for imaging in 1, 2, or 3 dimensions is thus made difficult or impossible. Furthermore, the detectors used for the FCS measurement must exhibit very high quantum yield and very good signal-to-noise ratio in order to be used for the photon counting needed for FCS measurements. Avalanche photo diodes (APDs) that possess a very small detector surface are primarily used, for which reason they are not suitable for spectral detection for which purpose they have been used to date (Leica, Zeiss).
A combination of fluorescence microscopy (e.g., confocal laser scanning microscopy; CLSM) and confocal fluorescence fluctuation spectroscopy (e.g., fluorescence correlation spectroscopy; FCS) permit the simultaneous imaging of the spatial distribution of fluorescent molecules in a sample and the dynamics of these molecules, e.g., as a result of movement processes such as directed transport or undirected diffusion. In existing systems and those known from the literature only one point at a time can be illuminated for fluorescence fluctuation analysis, and the associated fluorescence signal recorded and processed. In a raster-scan process, different points on the sample can be scanned successively.